The recent completion of the entire Saccharomyces cerevisiae genome sequence provides a unique opportunity to study the complete set of genes known to specify all aspects of this free-living eukaryote. We propose to develop new transposon-based technologies which will support genome-wide analysis of S. cerevisiae at two levels: i) large scale, systematic analysis of gene function, and ii) "directed" approaches within yeast labs aimed at understanding the genes and proteins involved in phenotypes of interest. The goals of the project are to i) construct and test new artificial transposons for functional genetic analysis in yeast, ii) to develop methods for transposon-based mutagenesis of individual library clones to facilitate gene identification and analysis, and iii) to develop efficient, transposon- based methods for comprehensive global mutagenesis of the yeast genome. These resources will be particularly useful with strains bearing complex sets of well-defined starting mutations, special genetic backgrounds, or custom modifications for genetic screens, where the comprehensive set of gene knockouts will not be useful. Another advantage is that these methods and reagents could be used in any lab to mutagenize a clone or strain of interest and mutants obtained are efficiently cross referenced with the yeast genome sequence. Because the cellular processes of yeast and higher eukaryotes are for the most part highly conserved, and involve highly conserved gene homologs, studies aimed at understanding the complete set of genes specifying this organism will lead to a greater understanding of human diseases such as cancer.